Multiplex display polymerase chain reaction amplifies and resolves related sequences sharing a single moderately conserved domain.
We present a technique, multiplex display polymerase chain reaction (MD-PCR), that amplifies and resolves coding sequences from messenger RNAs sharing only a single moderately conserved domain encoding eight or nine amino acids. The technique, a form of single-sided PCR, allows detection of known and novel genes in a family by using one degenerate primer complementary to a gene family-specific domain. A second common primer is complementary to an oligonucleotide ligated to a nearby restriction enzyme cleavage site. Uniquely, restriction enzyme digestion of single-stranded cDNA, a technique never previously performed to useful advantage, is used to increase the specificity and sensitivity of the technique. Up to several hundred bases of coding sequence are amplified simultaneously from many (potentially from all) genes in a specific family, yielding products of different sizes from different genes, and allowing amplified products to be resolved electrophoretically. Typically, more than 50% of the amplified sequences are from the targeted gene family and many of the amplified products are novel sequences. mRNAs representing less than 1 in 100,000 messages can be detected. The method allows the focused yet open-ended examination of genes in families known to be important in both normal cellular homeostasis and the etiology of many diseases.
Peale
F V
FV
Department of Pathology, University of Washington, Seattle 98195, USA. fpeale@u.washington.edu
Mason
K
K
Hunter
A W
AW
Bothwell
M
M
D000818:Animals; D002642:Chick Embryo; D017124:Conserved Sequence; D015252:Deoxyribonucleases, Type II Site-Specific; D005801:Genes, Homeobox; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D015588:Observer Variation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid
D011505:Protein-Tyrosine Kinases; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases; C024403:GGCC-specific type II deoxyribonucleases